Investigation of biotransformation of selenium in plants using spectrometric methods

Spectrochimica Acta Part B 130 (2017) 7–16

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Investigation of biotransformation of selenium in plants using spectrometric methods☆ Anna Ruszczyńska ⁎,1, Anna Konopka 1, Eliza Kurek, Julio Cesar Torres Elguera, Ewa Bulska University of Warsaw, Faculty of Chemistry, Biological and Chemical Research Centre, Zwirki i Wigury 101, 02-093 Warszawa, Poland

a r t i c l e

i n f o

Article history: Received 30 June 2016 Received in revised form 31 January 2017 Accepted 3 February 2017 Available online 04 February 2017 Keywords: Selenium speciation HPLC-ICP-MS HPLC-ESI-Orbitrap-MS/MS

a b s t r a c t The aim of this research was to study the processes of biotransformation of selenium in plants such as garlic, radish sprouts and sunflower sprouts via identification of selenium-containing compounds as metabolites of inorganic selenium using mass spectrometry. Speciation analysis of selenium in extracts from plant samples was performed with the use of hyphenated high performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method. Matching the retention times of sample compounds with standards allowed identification of Se-methyl-selenocysteine, selenomethionine, γ-glutamyl-Se-methylselenocysteine and 82 Se signals which couldn't be identiinorganic SeO2− 3 . However, registered chromatograms included additional fied due to the lack of standards. Qualitative analysis of unknown compounds was achieved using high-resolution mass spectrometer equipped with mass analyzer Orbitrap coupled to high performance liquid chromatography. Since selenium has six stable isotopes of different abundance in nature, mass spectra of have a very characteristic isotopic pattern. In order to elucidate the structure of unknown Se compounds, selected ions were subjected to the fragmentation. Following selenocompounds were identified an inorganic selenium metabolites in garlic, sunflower sprouts and/or radish sprouts: selenohomolanthionine, Se-methyl-selenocysteine, selenomethionine, selenomethionine oxide, deaminohydroxy-selenohomolanthionine, N-acetylcysteine-selenomethionine, γ-glutamyl-Se-methylselenocysteine, methylseleno-Se-pentose-hexose, Se-methyl-selenoglutathione, 2,3-dihydroxy-propionylselenocysteine-cysteine, methyltio-selenoglutathione, 2,3-dihydroxypropionyl-selenolanthionine and two Secontaining compounds with proposed molecular formula C10H18N2O6Se and C10H13N5O3Se. Moreover, the structure was proposed for one selenocompound found in sunflower sprouts which has not been reported so far. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Concern for human health increases the interest in research studies related to the biotransformation of macro- and microelements in living organisms. Selenium is one of essential trace elements and since the pioneer publication [1] reporting its beneficial role to the organisms a lot of effort has been taken to find out and understand the role of this element in physiological and pathological processes. Inter alia seleniumcontaining compounds have been recognized as antioxidant or chemo-preventive agents in cancer therapy [2–5]. Plants are the main source of selenium for animals and human and they transform most of the inorganic selenium species present in soils into organic selenium compounds thus making them easily accessible to animal organisms. Unfortunately there is a limited number of selenophilic plants and additionally a lack of selenium in European soils [6]. Therefore, together with growing interest in pharmaceutical ☆ Selected paper from the European Symposium on Atomic Spectrometry (ESAS 2016), Eger, Hungary, 31 March–2 April 2016. ⁎ Corresponding author. E-mail address: [email protected] (A. Ruszczyńska). 1 Authors equally contributed to the work

http://dx.doi.org/10.1016/j.sab.2017.02.004 0584-8547/© 2017 Elsevier B.V. All rights reserved.

supplementation of various key microelements a lot of attention is paid to the nutritional supplementation of selenium and the design of functional food [7,8]. The knowledge of selenium speciation in plants is crucial for understanding the metabolic pathways of this element as well as for demonstrating the beneficial role of these plants in order to find the ways for human diet enriched with selenium. The bioavailability of selenium is determined primarily by its chemical form [9] and in less extent by its total concentration, physiological conditions of species or other constituents of the diet [3,10]. Although selenium is not considered as an essential plant nutrient, more selenium species have been identified in plants than in animals, e.g.: inorganic compounds as selenate Se(VI) and selenite Se(IV), organic forms: selenomethionine (SeMet), selenocysteine (SeCys), Se-methyl-selenocysteine (Se-methyl-SeCys), γ-glutamyl-Se-methyl-selenocysteine (γ-glutamyl-Se-methyl-SeCys), selenoproteins. Some plants also volatilise selenium as dimethyl-selenide or dimethyl-diselenide [11–15]. Plants transport most of inorganic selenium (selenite and selenate) from soil by roots to the chloroplasts where it is transformed by the sulphur–assimilation pathway into selenoamino acids [16,17]. Some of the products or their intermediates are well known while the part of it hasn't been still identified. Their identification may have a great impact

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on the knowledge about selenium metabolic pathways like in case of selenohomolanthionine identified in Japanese pungent radish [14]. Selenium compounds in biological samples are identified and determined with the use of separation techniques coupled to sensitive detection methods: ion chromatography coupled to inductively coupled plasma mass spectrometry method (IC-ICP-MS) [18], high performance liquid chromatography coupled to ICP-MS (HPLC-ICP-MS) [19–24], size exclusion chromatography coupled to ICP-MS (SEC-ICP-MS), liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) [25–27]. In most biological materials different extraction procedures of selenium compounds are applied using various extractants, predominantly water [14,28–31] and enzymes [32–35]. Gas chromatography is used in case of volatile selenocompounds [36]. Usefulness of tandem mass spectrometry (MS/MS) with soft ionization techniques (e.g. electrospray, ESI) in identification and structural characterization of molecules at trace levels in complex biological matrices was demonstrated in the end of XXth century for the first time and is popularity is still growing [26]. At the beginning metabolomic studies were based on low resolution instrumentation (quadrupole, ion trap) [37,38] which with time had been replaced with high resolution systems (time-of-flight, Orbitrap) and combined in MS/MS tandem systems. Large diversity of small molecule compounds as well as a wide range of their concentration in natural samples is problematic in case of use conventional low resolution MS analyzers. An Orbitrap analyzer used for metabolite profiling providing very accurate mass-to-charge (m/z) measurements (below 1 ppm) at high resolving power (500 000) without using strong magnetic field had become popular since the first presentation [39,40]. The number of possible molecular structures which can be elucidated for masses measured by Orbitrap but only these ones containing elements with biological impact (C, H, O, N, P and S) is decreased to minimum due to its high accuracy of m/z ratio measuring capability. Presence of two analyzers provides the opportunity to register the fragmentation spectra thus allowing the identification of molecular structures without using standards which are limited in case of selenocompounds. Many studies on the speciation of Se in plants regarding vegetables such as soybean [11], mustard [12], radish [14], onion [23], garlic [32], broccoli [33] or kale [41] have been reported. Most of the selenium accumulating vegetables needs preliminary selenium fertilization of soil with selenite or selenate which may lead to the environmental pollution. Contrary to that, sprouts are germinated in hydroponic closed systems. In case of vegetables, which have to be boiled prior to consumption, some important selenium compounds can be lost by extraction into the water or transformation into other compounds. In contrast, sprouts can be consumed raw without any temperature treatment. Moreover, selenium enriched edible sprouts are a very interesting plant material containing significant amount of isoflavones, vitamins, minerals and folic acid and additionally there are known as selenium accumulators. Rather limited literature concerning selenium speciation in sprouts has reported the presence of SeMet [7,21,42–45], Se-methylSeCys [21,42,44], SeCys [7,43], γ-glutamyl-Se-methyl-SeCys [21,42– 44] as well as unidentified selenium compounds. The aim of this study was to investigate the speciation of Se in garlic, sunflower sprouts and radish sprouts, all grown in selenium enriched conditions, with the special focus on the identification of selenocompounds present in sprouts which have been not reported so far. We present the complete analytical methodology for identification of Se containing compounds using HPLC-ICP-MS and HPLC-ESIOrbitrap-MS/MS in case of lack of standards.

Hettich Zentrifugen (Germany) for separation of supernatants from plant tissue residue was used. Isotope specific detection was achieved using quadrupole mass spectrometer with inductively coupled plasma ionization, ICP-MS, (Nexion 300D, Perkin Elmer Sciex, USA) equipped in quartz cyclonic spray chamber, Meinhard nebulizer and platinum sampler and skimmer cones. The working conditions of spectrometer were optimized daily in order to obtain the maximal sensitivity and stability as well as the lowest level of oxides and double charged ions. The isotope 82Se with 8.7% of natural abundance was used for quantification due to the interferences coming from plasma gas (40Ar40Ar) for the most abundant isotope 80Se. An Agilent 1260 Infinity high performance liquid chromatography (Agilent Technologies, USA) was used with anion exchange Hamilton PRP-X100 (250 mm × 4.6 mm, particle size 10 μm) column from Hamilton (USA) and coupled with polyetheretherketone (PEEK) tubing to Elan 6100 DRC ICP-MS system (Perkin Elemer Sciex, Canada) for HPLC-ICP-MS analysis in plant extracts. A vacuum centrifuge (Eppendorf, Germany) was used for concentration of the fractions collected from an anion exchange liquid chromatography according to the 82Se peak detection. An Agilent 1290 Infinity high performance liquid chromatography system (Agilent Technologies, USA) equipped with the Porous Graphitic Carbon (PGC) Hypercarb column (150 mm × 4.6 mm, particle size 5 μm) purchased from Thermo Scientific was coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, USA). 2.2. Reagents, solutions, materials and samples Analytical reagent grade chemicals purchased from Sigma Aldrich (Germany), Baker (Holland), Merck (Germany) and water (18.2 MΩ cm) obtained with Milli-Q system (Millipore, Bedford, MA) were used throughout. The standards of Se compounds were purchased from Sigma-Aldrich. Multi elemental ICP-MS standard was obtained from Merck and Selenium Enriched Yeast certified reference material (SELM 1) from National Research Council Canada. Working solutions were obtained by dilution with HNO3 acidified deionized water as necessary. Water and acetonitrile of LC MS grade (Baker, USA) were used for HPLC-ESI-MS/MS experiments. Formic acid was purchased from Sigma Aldrich. Samples of sunflower sprouts and radish sprouts were germinated with tap water (control) and 40 mg of Se L−1 (sunflower sprouts) and 20 mg of Se L−1 (radish sprouts) in the form of sodium selenite −1 of (SeO2− 3 ). Garlic was grown in soil cultivated with 350 mg of Se L the same inorganic Se form. After harvesting all plants were air dried in the temperature below 40 °C, grounded and stored at 4 °C. 3. Procedures 3.1. Extraction The samples in the amount of 0.1 g were extracted with 5 mL water with addition of 0.02 g lipase (lipase from Candida rugosa, Sigma Aldrich) and 0.02 g protease (protease from Streptomyces grisens type XIV, Sigma Aldrich). Extraction was conducted in 37 °C over 21 h supported by magnetic stirring. The supernatant was separated from the residue by centrifugation for 20 min at 1000 rpm (centrifuge model EBA-20 from Hettich Zentrifugen) and filtered through 0.45 μm membrane filters. The extracts were kept at −15 °C temperature before the chromatography separation was executed. Extraction yield determination was performed after dilution of extracts in 1% HNO3.

2. Materials and methods 3.2. Total Se determination 2.1. Apparatus The microwave system Ultra Wave (Milestone, Italy) was used for digestion of samples and sample extracts. A centrifuge EBA-20 from

A volume of 1 mL of sample extracts were digested with 5 mL HNO3 and H2O2 using the following program: 20 min up to 250 °C and 15 min at 250 °C. After cooling down the digests were diluted with 1% HNO3

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and analyzed by ICP-MS. Quantitation was achieved by 5 point external calibration (standards from 1 μg L−1 to 100 μg L−1, correlation coefficient R2 = 0.9998, limit of linearity 105 μg L−1) and validated by the analysis of SELM-1 reference material. The obtained (1942 mg kg− 1 ± 99 mg kg− 1) and certified (2059 mg kg− 1 ± 64 mg kg−1) values were in agreement. The limit of detection for 82Se was 0.39 × 10−3 mg kg−1. 3.3. HPLC-ICP-MS analysis Mobile phases for anion exchange liquid chromatography were prepared by dissolving an appropriate amount of ammonium acetate in deionized water to obtain the required concentrations at pH = 4.7: i) 5 mmol L−1 (solvent A) and ii) 150 mmol L−1 (solvent B). The mobile phase flow rate was 1 mL min−1 at 22 °C. The solutions were filtered and degassed before the use. The injection volume was 100 μL. Compounds were eluted with the increasing linear gradient from 0%–100% of solvent B within 21 min. The chromatograms were obtained with 82 Se detection. In order to perform identification by ESI-MS/MS eluates were collected to vials at times corresponding to the retention times of previously registered selenium signals. The volumes of collected fractions were various depending on the selenium registered peak width.

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11 mg kg−1, respectively. The extraction efficiencies were calculated to be for sunflower sprouts, radish sprouts and garlic 64%, 48% and 62%, respectively. The total selenium determination for extracts was performed in order to select for succeeding speciation analysis extracts characterized by the highest selenium concentration thus increasing the probability of identification of low abundant selenium compounds. As well sprouts as garlic, which is one of the species in the onion genus, Allium, have ability to accumulate significant amounts of selenium and the obtained in extracts concentration values allowed for further speciation analysis which results are presented in Fig. 1. The applied chromatographic conditions demonstrated sharp peaks and good separation efficiency within 20 min. The registered retention times for standards were for Se-methyl-SeCys – 2.70 min, for SeMet – 4.70 min, for SeO23 − 9.25 min, for γ-glutamyl-Se-methyl-SeCys – 12.65 min and for SeO2− 4 - 15.15 min (Fig. 1a). At the time of 2.17 min a low intensity signal appeared described here as unknown compound. Matching the retention times of commercially available standards with peaks in plant extracts indicates the presence of SeMet and γglutamyl-Se-methyl-SeCys in all of them, Se-methyl-SeCys in garlic in both sprouts extracts. In no and radish sprouts extracts and SeO2− 3 case of real samples signal at the retention time of SeO24 − standard was registered. In all cases some unidentified peaks appeared. All

3.4. HPLC-ESI-MS/MS analysis Collected fractions were concentrated in a vacuum centrifuge to the dryness and re-suspended in 2% aqueous formic acid in a final volume of 23 μL. Samples were loaded directly into analytical column at the flow rate of 0.5 mL min−1 of 3% (v/v) solvent B (acetonitrile with 0.1% formic acid). The injection volume was 20 μL. Compounds were eluted from the column at the flow rate of 0.5 mL min−1 with the increasing linear gradient from 3%–50% of solvent B in 16 min or 26 min depending on the MS acquisition method. 0.1% aqueous formic acid was used as solvent A. The eluted compounds were ionized in the positive ion mode with a capillary voltage of 3.9 kV in the heated ion source HESI. The ion source parameters optimized on the total ion current TIC values were as follows: sheath gas flow 40 L min−1, auxiliary gas flow 15 L min−1, ion transfer tube temperature 350 °C and vaporizer temperature 300 °C. Survey scans were recorded with the Orbitrap mass analyzer at resolving power of 60 000 in the m/z range of 50–1000 and from each survey scan the top 10 most abundant singly charged ions (parent ions) were isolated for fragmentation by higher energy collision induced dissociation (HCD). The parent ions were isolated with the isolation window of 10 Da or 2 Da depending on the used conditions. The product ions were analyzed in the Orbitrap analyzer at the resolving power of 15 000. After fragmentation the masses were excluded for 30 s from further fragmentation. 3.5. HPLC-ESI-MS/MS data evaluation Survey scans were searched manually using Xcalibur 3.0 (Thermo Scientific, USA) for the presence of isotopic pattern characteristic for selenium-containing compounds. MS/MS spectra were interpreted manually in order to elucidate the structure of the unknown compounds. For every identified selenocompound the difference in ppm (δ) between its theoretical and experimental mass was calculated. The theoretical isotopic pattern of the unknown selenocompound, simulated using Xcalibur, was compared to the experimental one manually. 4. Results and discussion 4.1. Total selenium concentration and HPLC-ICP-MS analysis The total Se concentrations determined in extracts from sunflower sprouts, radish sprouts and from garlic were 73.6 mg kg− 1 ± 5.0 mg kg− 1, 156 mg kg−1 ± 8 mg kg−1 and 182 mg kg− 1 ±

Fig. 1. Chromatograms of 82Se signal obtained using HPLC-ICP-MS for analysis of a) standards and plant extracts: b) garlic, c) sunflower sprouts and d) radish sprouts.

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registered signals are described with order numbers for each plant extract: garlic (GF1–GF9), sunflower sprouts (SF1–SF8) and radish sprouts (RF1–RF8) which correspond to the collected fractions for further ESIMS/MS analysis and are indicated in grey fields in the Fig. 1. Depending on the stage of development and the species, plants may metabolise an inorganic selenium added to subsoil in the form of SeO2− 3 differently. It is worth to mention that in case of both kinds of sprouts (sunflower and radish) seven signals were registered at the same retention times: SeMet, SeO2− 3 , γ-glutamyl-Se-methyl-SeCys and unidentified marked as SF1 = RF1, SF4 = RF5, SF6 = RF6, SF7 = RF8. In case

of signals obtained for full-grown plant (garlic) at the same retention times were registered in both sprouts extracts for SeMet and γglutamyl-Se-methyl-SeCys only, in radish sprouts extract for Se-methyl-SeCys and in sunflower sprouts extract for unknown GF6 = SF5. Additionally, obtained signals for garlic extract had the highest intensity among all plant extracts which is in agreement with total selenium content determined in extracts. Due to the uncertainty of retention times the identification of obtained signals on the bases of comparison with commercially available standards has to be confirmed by further identification analysis.

Table 1 Selenocompounds detected in particular fractions and/or full extract of each plant by ESI-Orbitrap-MS/MS with the corresponding masses. m/zexp [M + H]+ Plant extract

Fraction and/or full extract

Identified compound

Full extract, m/ztheor [M + H]+ δ

Garlic

GF1 and full extract

Selenohomolanthionine

285.0348

Full extract

Se-methyl-selenocysteine

183.9871

GF2 and full extract

166.9606

GF3 and full extract

Se-methyl-selenocysteine — NH3 (neutral loss of ammonia in the ESI ion source) Selenomethionine

198.0028

GF3

Selenomethionine oxide

213.9977

GF3 and full extract

Selenomethionine oxide — H2O (neutral loss of water in the ESI ion source) – Deaminohydroxy-selenohomolanthionine

195.9871 – 286.0188

N-Acetyl-cysteine-selenomethionine

345.0023

GF7 and full extract

γ-Glutamyl-Se-methyl-selenocysteine

313.0297

GF8

Methylseleno-Se-pentose-hexose

407.0456

195.9876 2.55 ppm – 286.0191 1.05 ppm 345.0025 0.58 ppm 313.0300 0.96 ppm –

GF9

C10H19N2O6Se+

343.0403

–

Full extract

Se-methyl-selenoglutathione

370.0512

370.0515 0.81 ppm

SF1

Se-methyl-selenocysteine — NH3 (neutral loss of ammonia in the ESI ion source) Selenomethionine

166.9606

–

198.0028

–

–

198.0032 2.02 ppm –

2,3-Dihydroxypropionyl-selenocysteine-cysteine-alanine

448.0287

2,3-Dihydroxypropionyl-selenocysteine-cysteine

376.9916

Methyltio-selenoglutathione

402.0233

RF1

C10H14N5O3Se+

332.0256

RF2

Se-methyl-selenocysteine

183.9871

RF2 and full extract

Se-methyl-selenocysteine — NH3 (neutral loss of ammonia in the ESI ion source) Selenomethionine

166.9606

GF4, GF5 GF6 and full extract

Sunflower sprouts

SF2 and full extract SF3, SF4, SF5, SF6, SF7, SF8 Full extract

Radish sprouts

285.0350 0.70 ppm 183.9874 1.63 ppm 166.9608 1.20 ppm 198.0031 1.52 ppm –

448.0292 1.12 ppm 376.9922 1.59 ppm 402.0237 0.99 ppm

213.9977 195.9871

RF7

Selenomethionine oxide Selenomethionine oxide — H2O (neutral loss of water in the ESI ion source) γ-Glutamyl-Se-methyl-selenocysteine

332.0249 −2.11 ppm 183.9875 2.17 ppm 166.9610 2.40 ppm 198.0031 1.52 ppm – –

313.0297

–

RF4, RF5, RF6, RF8 Full extract

– 2,3-Dihydroxypropionyl-selenolanthionine

– 345.0196

2,3-Dihydroxypropionyl-selenocysteine-cysteine

376.9916

– 345.0201 1.45 ppm 376.9921 1.33 ppm

RF3 and full extract RF3

198.0028

Fraction, δ

Ref.

285.0340 [14,46–48] −2.81 ppm – [11,29,49] 166.9600 −3.60 ppm 198.0020 −4.04 ppm 213.9970 −3.27 ppm 195.9870 −0.51 ppm – 286.0180 −2.80 ppm 345.0010 −3.77 ppm 313.0290 −2.24 ppm 407.0440 −3.93 ppm 343.0390 −3.80 ppm –

[49,50] [29,46,48,51] [52] [46,48,50] – [11] [46,50] [11,29,47,49,50,53] [15] [11] [47,50,53]

166.9623 [49,50] 10.18 ppm 198.0022 [29,46,51] −3.03 ppm – – –

–

–

[46,48]

–

[46,54]

332.0231 – −7.52 ppm – [11,29,49]

313.0284 [11,29,47,49,50,53] −4.15 ppm – – – [47,48]

166.9602 −2.40 ppm 198.0022 −3.03 ppm 213.9971 195.9869

–

[49,50] [29,46,48,51] [52] [46,48,50]

[46,48]

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4.2. Selenium speciation using HPLC-ESI-Orbitrap-MS/MS In order to confirm the identity of the selenium containing compounds identified by HPLC-ICP-MS and to identify the unknown selenocompounds high performance liquid chromatography coupled to electrospray tandem mass spectrometer HPLC-ESI-Orbitrap-MS/MS was applied. Selenocompound fractions were collected manually according to the retention times of selenium peaks registered during HPLC-ICP-MS analysis carried out for all studied extracts as indicated at Fig. 1(b–d). Next, each fraction was concentrated in a vacuum centrifuge and subjected to HPLC-ESI-Orbitrap-MS/MS analysis. Additionally, full extracts of all studied plants were analyzed by HPLC-ESI-OrbitrapMS/MS. Both, survey scans and fragmentation spectra were recorded by Orbitrap analyzer ensuring the very accurate measurement of the mass to charge ratio of molecular ions below 5 ppm. As a result of HPLC-ESI-Orbitrap-MS/MS analyses the identity of all selenium metabolites, previously identified by HPLC-ICP-MS, was confirmed and additional selenium compounds were identified. Identified compounds found in the collected fractions and/or in the full extracts for all studied plants are listed in Table 1. The following methodology used for searching and identification of unknown selenium compounds is briefly showed for an example of Se-methyl-Se-glutathione identification. The registered HPLC-ESIOrbitrap-MS chromatograms (survey scans) were searched manually for the presence of the isotopic pattern characteristic for chemical compounds containing selenium. Due to the fact that selenium has six stable isotopes of different abundance in the nature, its chemical compounds are easily visible in the MS survey scan because of its very characteristic isotopic pattern. The survey scan with such isotope pattern for registered selenocompound of m/z 370.0512 is shown in the Fig. 2. Next, in order to check the chromatographic profile and retention time, the found mass to charge ratio was extracted from the registered total ion current (TIC) as an extracted ion choromatogramm (EIC) as presented in the Fig. 3. In order to elucidate the structure of found unknown selenium compound, its fragmentation MS/MS spectra should be evaluated. The molecular ion with m/z 370.0512 was isolated for fragmentation with two values of isolation window 2 Da and 10 Da. For narrower isolation window of 2 Da for the molecular ion only monoisotopic mass is included for the fragmentation whereas for the broader isolation window of 10 Da also ions with other isotopes of elements (isotopomers) are included for fragmentation. This results in a presence of characteristic isotopic Se pattern at the MS/MS spectra as well, which provides an

Fig. 3. HPLC-ESI-MS chromatogramms registered for garlic full extract: a) total ion current (TIC); b) extracted ion chromatogramm (EIC) for m/z 370.0512.

additional information about the presence of Se in the product ions. Such MS/MS spectra isolated with window of 2 Da and 10 Da are presented in the Fig. 4a and b. Careful evaluation of MS/MS spectra by matching the registered m/z of product ions with theoretical ones resulted in the structure elucidation of the unknown selenocompound. The fragment ions which belong to the structure of Se-methyl-Se-glutathione and matching the recorded MS/MS spectra are presented in the Fig. 4c. Once the chemical formula and the structure of the unknown selenium-containing compound is elucidated, its isotopic pattern recorded in

Fig. 2. MS survey spectrum registered with zoom of the m/z region 365–373 presenting the isotopic pattern characteristic for selenium-containing compound.

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Fig. 4. MS/MS spectrum for the ion of m/z 370.0512: a) registered with the isolation window of 2 Da; b) registered with the isolation window of 10 Da (product ions which are recognized as characteristic fragment ions of Se-methyl-Se-glutathione are marked in the circles); c) fragmentation pathway for Se-methyl-Se-glutathione.

MS survey scan (Fig. 2) is compared to the theoretical one, simulated in silico. Such comparison for the [M + H]+ ion for Se-methyl-Se-glutathione is presented in the Fig. 5. An HPLC-ICP-MS method allowed the identification of four most intense selenocompounds using available standards: Se-methyl-SeCys, in plant extracts by reSeMet, γ-glutamyl-Se-methyl-SeCys and SeO2− 3 tention time matching with standards which is in agreement with previously reported selenium speciation studies in sprouts [21,35,42–45] and garlic [32] extracts. However presented chromatogramms in extracts include several unidentified signals sometimes overlapping each other. Considering that some different compounds with similar properties can have congenial or even identical retention times as well as uncertain purity of chromatographic signals and their low intensity in real

samples, the formal identification of detected compounds with HPLCESI-Orbitrap-MS/MS was conducted. According to expectations the identity of most of identified with HPLC-ICP-MS compounds were confirmed during HPLC-ESI-MS/MS analysis either in full extracts and/or in particular fractions. In case of garlic full extract as well as particular fractions (GF2, GF3 and GF7) the presence of all identified with HPLC-ICP-MS compounds were confirmed: Se-methyl-SeCys, SeMet and γ-glutamyl-Se-methylSeCys. Additionally, in four out of six collected fractions six different selenocompounds were identified. Just for one selenium containing compound of measured m/z of 343.0393 only molecular formula C10H18N2O6Se was proposed. The selenocompound of very similar theoretical m/z value of 343.0403 ((δ = − 2.92 ppm in this study) was

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Fig. 5. Isotopic pattern recorded for Se-methyl-Se-glutathione: a) experimental, b) theoretical simulated in silico.

reported by Ouerdane et al. [11] and has been recognized as Se-glucosyl-Se-allyl-N-hydroxy-selenourea. The MS/MS spectra recorded in the course of our study did not confirm such chemical structure of this selenocompound. Concerning this further studies are in progress. During this study only garlic belongs to mature plants, therefore it was expected that in sprouts extracts different selenocompounds would be found. Even though during HPLC-ICP-MS analysis signal marked as GF1 was considered as the same compound as SF1 and RF1, as well as signal GF6 with SF5. The result of identification by HPLCESI-MS/MS analysis showed different selenocompound for each plant extract in the first mentioned fractions. In second case of GF6 considered as the same as SF5 nothing was detected in sunflower sprouts fractions probably due to the very low concentration of the present selenocompounds. Since ICP-MS is a very sensitive method and very tolerant to the complex matrix, some low abundant selenium containing compounds can be still recorded, whereas they are not observed in ESI-MS because of e.g. ion suppression effect, which considerably increases its limit of detection. Signal present at HPLC-ICP-MS chromatogramm at fraction GF6 consisted of two different selenocompounds identified by HPLC-ESI-MS/MS as deaminohydroxyselenohomolanthionine and N-acetyl-cysteine-selenomethionine. That clearly confirms the need of additional identification by ESI-MS/MS. In case of extracts of different plants (sunflower and radish) but at the same phase of development it was expected to find more similarities in selenium speciation. The results of identification analyses were more problematic due to the lower concentration of selenocompounds in sprouts, especially in case of sunflower sprouts, characterized by the lowest total selenium concentration determined by ICP-MS. Probably therefore, in fractions from SF3 to SF8 and in RF4, RF5, RF6 and RF8 none of selenocompound was identified using HPLC-ESI-MS/MS. At the same time in full extracts of sunflower sprouts and radish sprouts

compounds not registered during HPLC-ICP-MS analysis were identified. In radish sprouts one selenocompound of measured m/z of 332.0249 in the full extract was found. It is probably 5′selenoadenosine with the theoretical mass of [M + H]+ = 332.0256 (δ = −2.11 ppm in this study) previously reported in the literature [46,50]. Since in our study the MS/MS spectrum was not recorded for this m/z value, the proper accurate verification of the chemical structure based on product ions could not be performed. Further studies need to be done. One of selenium containing unknown compound found in sunflower sprouts extract with m/z of 448.0292 seemed to be 2,3dihydroxypropionyl-selenohomocysteine-cysteine-glycine reported previously by Arnaudguilhem et al. [46] to be present in selenized yeast. However, in the registered MS/MS spectrum (shown in Fig. 6a) and b)) the product ion of m/z 269.9875 (theoretical) characteristic for the 2,3-dihydroxypropionyl-selenohomocysteine (Fig. 6c) was not present and therefore this compound cannot be identified as 2,3dihydroxypropionyl-selenohomocysteine-cysteine-glycine. On the other hand, the signal for m/z value of 255.9724 was found which is characteristic for the 2,3-dihydroxypropionyl-selenocysteine (m/ ztheor. = 255.9719, δ = 1.95 ppm). On the basis of other product ions the seleno-compound of m/z of 448.0292 was identified as 2,3dihydroxypropionyl-selenocysteine-cysteine-alanine (m/ztheor. = 448.0287, δ = 1.56 ppm) as shown in the Fig. 6d. The MS/MS spectra and the proposed structure of new selenocompound are shown in the Fig. 6. This selenocompound has not been reported so far to our best knowledge. 5. Conclusions The use of an HPLC-ICP-MS and HPLC-ESI-MS/MS allowed for the speciation of selenometabolites in extracts of different plants: garlic, sunflower sprouts and radish sprouts. The presence of three

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Fig. 6. MS/MS spectrum for the ion of m/z 448.0292: a) registered with the isolation window of 2 Da; b) registered with the isolation window of 10 Da (product ions which are recognized as characteristic product ions of 2,3-dihydroxypropionyl-selenocysteine-cysteine-alanine are marked in the circles); c) chemical structure of 2,3-dihydroxy-propionylselenohomocysteine with product ions marked [39]; d) fragmentation pathway for 2,3-dihydroxypropionyl-selenocysteine-cysteine-alanine.

selenocompounds for which standards were commercially available was confirmed by HPLC-ICP-MS matching the retention times of registered selenium signals with those for standards. Due to the possibility of overlapping signals and inaccuracy of retention times during HPLCICP-MS method a formal identification by molecular ESI-MS/MS should be considered in each case. In a course of this study a number of other compounds containing selenium were identified by HPLC-ESI-MS/MS analysis: selenohomolanthionine, Se-methyl-selenocysteine, selenomethionine,

selenomethionine oxide, deaminohydroxy-selenohomolanthionine, Nacetylcysteine-selenomethionine, γ-glutamyl-Se-methyl-selenocysteine, methyl-seleno-Se-pentose-hexose, Se-methyl-selenoglutathione, 2,3-dihydroxy-propionyl-selenocysteine-cysteine, methyltio-selenoglutathione and 2,3-dihydroxypropionyl-selenolanthionine. In case of two compounds only a molecular formula was proposed: C10H18N2O6Se and C10H13N5O3Se. Moreover, one structure was proposed for 2,3-dihydroxypropionylselenocysteine-cysteine-alanine found in sunflower sprouts, which has not been reported so far.

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Acknowledgments The study was carried out at the Biological and Chemical Research Centre, University of Warsaw established within the project co-financed by European Union from the European Regional Development Fund under the Operational Programme Innovative Economy 2007–2013. This project was financed in the framework of grant entitled: Investigation of chemical and biological processes of selenium biotransformation in selenophilic plants and probiotic bacteria towards their application as functional food attributed by the National Science Centre, Poland (2012/05/ B/ST4/01219). References [1] K. Schwarz, C.M. Foltz, Selenium as an integral part of factor 3 against dietary necrotic liver degeneration, J. Am. Chem. Soc. 79 (1957) 3292–3293. [2] M.P. Rayman, The importance of selenium to human health, Lancet 356 (2000) 233–241. [3] S.J. Fairweather-Tait, Yongpin Bao, M.R. Broadley, R. Collings, D. Ford, J.E. Hesketh, R. Hurst, Selenium in human health and disease, Antioxid. 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